Dsrna-based gmo-free weed management tool for sustainable amaranthus hybridus control

Overview

This solution offers a GMO-free approach to managing the invasive and herbicide-resistant weed Amaranthus hybridus by exploiting the plant's natural RNA interference (RNAi) machinery. The technology uses double-stranded RNA (dsRNA) molecules designed to target the acetolactate synthase (ALS) gene, a well-established herbicide target, to achieve sequence-specific gene silencing. By providing an alternative to chemical herbicides, this tool addresses growing concerns over herbicide resistance, environmental contamination, and the sustainability of crop production systems.

Technical specifications

Core technology:

• Three exogenous dsRNA molecules targeting distinct regions of the endogenous ALS transcript in A. hybridus
• ALS is selected as the target because it is an intronless, single-copy gene, making it highly suitable for RNAi-based silencing
• dsRNAs are synthesized in vitro via T7 RNA polymerase transcription from linearized L4440 plasmid DNA containing two converging T7 promoter regions flanking the cloned sequence
• Complementary single-stranded RNAs are annealed at equimolar concentrations to form stable dsRNA molecules

Planned validation parameters:

• Dose-response testing across a range of dsRNA quantities (1 µg to 10-fold serial increases)
• Comparison of surfactant formulations (Silwet vs. Trend) for optimal delivery
• Evaluation of multiple application methods, including agro-infiltration, spraying, and nanocarrier-based delivery
• Monitoring of phenotypic variations in treated versus untreated plants
• Molecular analysis of small RNA (sRNA) profiles, ALS gene expression levels, and expression of key genes involved in the post-transcriptional gene silencing (PTGS) pathway

Technology readiness level

The technology is at an early-to-mid stage of development. A preliminary study has successfully completed the in vitro synthesis of dsRNA targeting the A. hybridus ALS gene. Future validation will be carried out through a collaboration between the National Research Council (CNR) and the University of Padova, supported by a one-year student scholarship. The next phase will focus on optimizing delivery methods and confirming effective post-transcriptional silencing of the ALS gene in planta, with parallel molecular characterization to validate the RNAi mechanism.